Project Summary/Abstract The post-translational modification of histone proteins is now established as an important mechanism for regulating gene expression in eukaryotic cells. However, a structural and mechanistic understanding of how the histone modification enzymes function on their nucleosome substrate is lacking. This shortcoming limits interpretation of the wealth of genetic, genomic and biochemical data available, and it hampers development of new therapeutics that target the many chromatin enzymes associated with human diseases including cancer. We are focused on addressing these deficiencies through structure determination of histone modification enzymes bound to their physiological nucleosome substrate. We propose to determine X-ray crystal structures of histone modification enzymes associated with human diseases in complex with the nucleosome, using established and novel approaches to crystallize these complicated, multicomponent protein/DNA complexes. We will complement our crystallographic efforts with use of cryoelectron microscopy for three-dimensional structure determination of chromatin enzyme/nucleosome complexes. We also plan to use cryoelectron microscopy to tackle structure determination of native, megadalton sized histone modification enzyme complexes.